Beyond Gene Inactivation: Evolution of Tools for Analysis of

Subscriber access provided by Yale University Library

Review

Beyond Gene Inactivation: Evolution of Tools for Analysis of Serotonergic Circuitry Cornelia Hainer, Valentina Mosienko, Stella Koutsikou, Jonathan J. Crook, Bernd Gloss, Sergey Kasparov, Bridget M Lumb, and Natalia Alenina ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00045 • Publication Date (Web): 01 Jul 2015 Downloaded from http://pubs.acs.org on July 3, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Beyond Gene Inactivation: Evolution of Tools for Analysis of Serotonergic Circuitry Cornelia Hainer1,*, Valentina Mosienko2,*,#, Stella Koutsikou2, Jonathan J. Crook2, Bernd Gloss3, Sergey Kasparov2, Bridget M. Lumb2, Natalia Alenina1,4 1

Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany

2

University of Bristol, Bristol, United Kingdom

3

National Institute of Environmental Health Science, Durham, NC 27709, USA

4

Institute of Translational Biomedicine, St. Petersburg State University, St. Petersburg,

Russia *equal contribution #

corresponding author

Corresponding author: Valentina Mosienko Medical Science Building University Walk, BS8 1TD, Bristol United Kingdom Tel: +441173312222 Email: [email protected]

Short running title: New tools to manipulate the serotonergic system

Key words: serotonergic system, Cre recombinase, optogenetics, Pet1, SERT, TPH2 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In the brain, serotonin (5-hydroxytryptamine, 5-HT) controls a multitude of physiological and behavioral functions. Serotonergic neurons in the raphe nuclei give rise to a complex and extensive network of axonal projections throughout the whole brain. A major challenge in the analysis of these circuits is to understand how the serotonergic networks are linked to the numerous functions of this neurotransmitter. In the past, many studies employed approaches to inactivate different genes involved in serotonergic neuron formation, 5-HT transmission or metabolism. Although these approaches have contributed significantly to our understanding of serotonergic circuits, they usually result in life-long gene inactivation. As a consequence compensatory changes in serotonergic and other neurotransmitter systems may occur and complicate the interpretation of the observed phenotypes. To dissect the complexity of the serotonergic system with greater precision, approaches to reversibly manipulate subpopulations of serotonergic neurons are required. In this review, we summarize findings on genetic animal models that enable control of 5-HT neuronal activity or mapping of the serotonergic system. This includes a comparative analysis of several mouse and rat lines expressing Cre or Flp recombinases under Tph2, Sert, or Pet1 promoters with a focus on specificity and recombination efficiency. We further introduce applications for Cre-mediated cell-type specific gene expression to optimize spatial and temporal precision for the manipulation of serotonergic neurons. Finally, we discuss other temporally regulated systems, such as optogenetics and DREADD approaches to control 5-HT neuron activity.

ACS Paragon Plus Environment

Page 2 of 38

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Contents 1. Introduction ............................................................................................................................... 4 2. Targeted expression of Cre and Flp recombinases in serotonergic cells .......................... 5 2.1. Cre recombinase expression driven by the Sert promoter ........................................... 7 2.1.1. Knock-in SERT-Cre ................................................................................................... 7 2.1.2. BAC-SERT-Cre .......................................................................................................... 8 2.2. Recombinase expression driven by the Pet1 promoter ................................................ 8 2.2.1. ePet1-Cre ................................................................................................................... 8 2.2.2. ePet-Flpe .................................................................................................................... 8 2.2.3. Pet1210-Cre ................................................................................................................. 8 2.2.4. BAC-Pet1-Cre ............................................................................................................ 9 2.2.5. BAC-Pet1-CreERT2................................................................................................... 9 2.3. Cre recombinase expression driven by the Tph2 promoter ........................................ 10 2.3.1. BAC-TPH2-CreERT2 ............................................................................................... 10 2.3.2. BAC-TPH2-CreERT2 ............................................................................................... 10 2.3.3. BAC-TPH2-iCreERT2 .............................................................................................. 10 3. Targeting defined serotonergic subpopulations .................................................................. 11 3.1 Dual recombinase strategies ...................................................................................... 11 3.2. Viral mediated gene transfer ...................................................................................... 12 3.2.1. Anterograde transport .............................................................................................. 13 3.2.2. Retrograde transport................................................................................................ 13 3.2.3. Transsynaptic transport ........................................................................................... 14 4. Temporal control of gene expression within the serotonergic system .............................. 15 4.1. Inducible Cre-expressing mouse lines .......................................................................... 15 4.2 Tetracycline-sensitive systems ....................................................................................... 15 4.3 DREADDs ......................................................................................................................... 16 4.4. Optogenetics ................................................................................................................... 17 Summary ..................................................................................................................................... 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 38

1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine that acts as a neurotransmitter in the central nervous system (CNS) and as an autacoid in the periphery. Serotonin synthesis in the CNS is restricted to serotonergic neurons, which originate from the raphe nuclei of the brain stem and extend vast axonal projections into different areas of the brain and spinal cord. Its synthesis is a two-step reaction. Tryptophan hydroxylase (TPH) 2 catalyzes the first, rate-limiting step in the CNS, in contrast to TPH1, which is active in the periphery.1-3 Tph2 is expressed exclusively in serotonergic neurons within the brain and in the enteric nervous system in the gut but not in other peripheral organs or neuronal cell types.2-6 Serotonin packaging into vesicles is mediated by vesicular monoamine transporter (VMAT) 2, which is expressed in monoaminergic neurons.7 Serotonin actions are mediated by its numerous receptors, that have different expression patterns within the CNS, including presynaptic locations.8,

9

For example, 5-HT1A autoreceptors, which are

expressed on soma and dendrites of serotonergic neurons, mediate inhibition of cell firing.10 Another important component of serotonergic neurons is serotonin transporter (SERT), which mediates 5-HT uptake.11 In the brain of adult animals, SERT is mainly expressed on 5-HT producing neurons of the raphe nuclei and astrocytes.12 During development, transient SERT expression in the brain is broader and includes nonserotonergic neurons in limbic, thalamic and retinal areas.13 Furthermore, SERT is located in a number of peripheral cells, such as platelets, adrenomedullary chromaffin cells, crypt cells in the gut, lung, liver, and testis.14,

15

SERT expression is therefore not

restricted to serotonergic neurons. Serotonergic neurons of the raphe nuclei are organized in groups, which are distinguished by their different anatomical location, axonal trajectories and physiological properties.16-18 The more rostrally located serotonergic cell groups, B6-B9, project to forebrain regions, whereas caudal serotonergic groups, B1-B5, project towards the spinal cord and brain stem.19 Although anatomically clustered, groups of serotonergic neurons are not homogenous

in

their

electrophysiological

properties

and

consist

of

different

20, 21

subpopulations, which are not fully characterized so far.

During brain development, serotonergic neurons arise from distinct units of the neural tube, named rhombomeres (r1-r3 and r5-r7).19 Developing 5-HT neuronal precursors along the rhombomeres express the PC12 ETS domain-containing transcription factor 1 (Pet1)


Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and LIM homeobox transcription factor 1 (Lmx1b) which are involved in serotonergic neuron differentiation. Lmx1b precedes Pet1 expression, and is also expressed in dopaminergic progenitors and neurons.22 Pet1 was thought to be a specific marker of differentiated post-mitotic serotonergic neurons.16,

23-25

However, more recent studies

indicated that Pet1 is also active in some non-serotonergic cells in adult and developing brain and in peripheral organs such as adrenal glands, the 5-HT-synthesizing enterochromaffin cells of the intestine, developing kidney, and pancreas.26, 27 The fact that development of serotonergic neurons, 5-HT synthesis, release and reuptake require expression of multiple specific genes was used for the analysis of serotonergic circuits. Mouse models were engineered to express different reporters specifically in serotonergic neurons, such as the transgenic ePet-EYFP28, the TPH2-GFP knock-in,29 and the TPH2-LacZ knock-in mouse lines.30 These models have been employed in various in vivo and in vitro studies to examine electrophysiological properties and morphology of 5HT neurons.28-30 To manipulate the serotonergic system, various strategies for enhancement or inhibition of neuronal activity have been used. Besides numerous pharmacological interventions, a number of studies employed genetic approaches to inactivate or overexpress different genes involved in serotonergic neuron formation, 5-HT transmission and metabolism.31-38 The behavior and physiological consequences of lifelong alterations in serotonergic signaling in the brain in these models were recently reviewed.39-42 The characterization of these genetic models contributed greatly to our understanding of serotonergic circuits. However, genetic approaches which result in life-long changes in gene expression (for example full knockouts or constitutive overexpression models) may induce compensatory mechanisms in other neurotransmitter systems, thus making it impossible to assess 5-HT function per se. Moreover, these models do not provide an opportunity to study functions of specific subpopulations of 5-HT neurons. In this review we summarize new tools which provide higher specificity and temporal control to study serotonergic circuitry.

2. Targeted expression of Cre and Flp recombinases in serotonergic cells To manipulate serotonergic neurons in the CNS of mice, many studies have made use of the standard Cre-LoxP approach for cell type-specific recombination. The Cre protein is a ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

site-specific DNA recombinase, that catalyses the recombination between two 34 bp long loxP sequences.43, 44 Cre-dependent recombination in conjunction with LoxP-flanked target genes or stop cassettes can inactivate or activate the expression of a gene of interest (GOI) respectively. Over the last decade, a number of transgenic and knock-in Creexpressing mouse lines have been generated. To direct transgene expression with the same developmental timing and spatial expression patterns as certain endogenous genes, bacterial artificial chromosomes (BACs) have also been widely used.45 BACs are DNA constructs, which are capable to host the DNA fragments of >350 kilobase pairs with a high degree of stability.46,

47

Therefore, they are suitable for inserting large promoter

regions including distant regulatory elements to drive Cre expression in specific cell populations.48, 49 Alternatively, knock-in approaches can be applied to control expression of the recombinase using the same machinery which controls expression of cell specific genes. In these cases the expression pattern is much more accurate and does not require time-consuming isolation and characterization of the promoter.50 Most importantly, the knocked-in transgene does not generate additional mutations at an unknown locus as classical randomly integrated transgenes do. Of note, during the knock-in approach one allele of the gene in which Cre is inserted will be disrupted leading to a heterozygous knockout of this gene unless the transgene is linked via a 2A-peptide sequence at the 3’end of the endogenous driver gene. For some genes, such as Tph2, the heterozygosity does not evoke behavioral alterations51, whereas for other genes, e.g., Sert, the deletion of one allele results in changes in neurochemistry and physiology (see below 2.1.1) and should be considered as an additional factor to be controlled for. Once a specific Cre line is successfully generated, the expression of recombinase can be functionally characterized by crossbreeding with Cre-dependent reporter strains, such as Rosa26loxPSTOP-lacZ or ROSA26loxP-stop-EYFP.52,

53

which start to express β-Galactosidase (β-

Gal) or EYFP, respectively, once Cre recombinase is active. Co-localization analysis of reporter expression and serotonergic markers is an important step in evaluating the specificity and confirming the efficiency of Cre-dependent recombination. In case of inducible Cre-ERT2 models, a chimeric protein consisting of a codon-optimized Cre recombinase fused to a triple mutant form of the human estrogen receptor (ERT2) is expressed in targeted cells. The mutant receptors no longer bind endogenous estradiol, but instead recognize the synthetic ligand Tamoxifen (TMX). Upon TMX administration, the recombinase gains access to the nuclear compartment and initiates recombination,54 thereby permitting temporal control of gene manipulation.


Page 6 of 38

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Another enzyme, flippase (Flp), which works similar to Cre and mediates DNA recombination between 34bp-long Flp recognition target (FRT) sites, has also been used in such approaches (reviewed in55). Available Cre and Flp transgenic mouse lines for the serotonergic system are summarized in Table 1, and discussed below in more detail.

2.1. Cre recombinase expression driven by the Sert promoter 2.1.1. Knock-in SERT-Cre Among the first published lines targeting specific neurotransmitter systems is the SERTCre mouse line, generated by knocking in Cre into the Slc6A4 locus (gene coding for SERT) by the group of R. Hen.50 In the raphe, 99% of cells which underwent recombination were TPH-positive, and nearly all serotonergic neurons were recombined. These results indicate a high recombination efficiency and specificity in the raphe of this SERT-Cre mouse line. This line has been widely used for different applications, including studies of heart development, effects of selective serotonin re-uptake inhibitors (SSRIs), sudden infant death syndrome, depression, addiction, development, and metabolism.56-60. For example, Narboux-Nême and colleagues disabled serotonergic transmission by crossing SERT-Cre to VMAT2fl/fl mice which led to a major (~95%) depletion of central 5HT, but not other monoamines, through excision of the VMAT2 in SERT-positive cells.61 However, this SERT-Cre line has several limitations. Firstly, due to the disruption of the Slc6A4 this SERT-Cre mouse represents a heterozygous knockout resulting in a mild behavioral and biochemical phenotype50 and therefore, proper controls (optimally SERTCre+ GOIfl/fl versus SERT-Cre+ GOIwt/wt) should be used in experiments. Another limitation of this line is Cre recombination occuring in non-serotonergic neurons of the thalamus, the cingulate cortex, and the CA3 region of the hippocampus (summarized in the Table1) due to transient SERT expression in these regions during early development.62 Furthermore, the SERT-Cre line is prone to germ-line transmission of the recombined allele, due to SERT-driven Cre expression in male germ cells.59 This may lead to the generation of a total, instead of a cell-type specific, knockout. By crossing SERT-Cre and ObRbfl/fl mice the group of G. Karsenty inactivated the leptin receptor b (LepRb) in serotonergic neurons.30 Based on the experiments performed in these mice they postulated that leptin regulation of appetite occurs by inhibition of 5-HT synthesis in the brain. However, a detailed analysis by other group could not confirm the expression of LepRb in serotonergic neurons and it



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

was supposed that the phenotypes, observed by Karsenty group were most probably resulting from total instead of conditional LepRb knockout59.

2.1.2. BAC-SERT-Cre Three BAC-SERT-Cre transgenic mouse lines were generated in frames of GENSAT project (http://www.gensat.org/index.html).49 In 2 founder lines (E33/E35) Cre expression was restricted to adult serotonergic neurons, whereas in the third line (E124) Cre expression was also observed in corticothalamic and thalamocortical neurons.49,

63

Moreover, in the E33 and E124 lines of BAC-SERT-Cre mice, Cre expression was observed in the developing visual system64 and the upper layers of the barrel cortex,65 respectively. These findings confirm SERT expression by non-serotonergic neurons during development, as shown earlier.13,

62

Taken together, BAC-SERT-driven Cre expression

targets all serotonergic neurons, however, ectopic SERT expression during development and developmental recombination in transgenic SERT-Cre lines must be considered.

2.2. Recombinase expression driven by the Pet1 promoter 2.2.1. ePet1-Cre Using a 40kb-long DNA region upstream of the Pet1 gene containing an enhancer essential for proper Pet1 expression, the laboratory of E. Deneris generated an ePet1-Cre line which enables Cre-mediated recombination restricted to all 5-HT neurons.28 Within the CNS, no ePet activity was detected outside the serotonergic system, however the analysis of LacZ expression in the periphery of R26RePet-Cre mice revealed Cre-dependent recombination in some cells of skin, gut, pancreas, and cardiac neural crest.

2.2.2. ePet-Flpe A Flp recombinase driver line, ePet-Flpe, was generated in the laboratory of S. Dymecki66 with the same ePet construct used by the Deneris’ group previously to generate the ePetCre line.28 In the raphe of ePet-Flpe mice, the activity of Flp is high and mediates recombination in most 5-HT raphe neurons.66

2.2.3. Pet1210-Cre To control somatic recombination in the Pet1 lineage more reliably, the group of M. Pasqualetti generated mice expressing Cre under control of a large 210kb-long fragment of the Pet1 genetic locus, including all putative regulatory regions.26 Pet1210-Cre transgenic ACS Paragon Plus Environment

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mice revealed the existence of non-serotonergic neuronal populations, which express Pet1 within the raphe, mostly in the B8 and B9 cell groups. Cre expression was also detected in embryonic kidney and pancreas, demonstrating that Pet1210-Cre mice are useful to study rather Pet-1 expressing lineages other than 5-HT neurons in particular (Table 1). Based on these findings, the specificity of other Pet-1 driven Cre lines and reporters, such as ePet1EYFP,28 should be reevaluated in these non-serotonergic neuronal populations and peripheral tissues.

2.2.4. BAC-Pet1-Cre Another BAC-based Pet1-Cre mouse line was generated by the group of Y.Q. Ding67 by insertion of the Cre coding sequence into the Pet1 locus. After crossing with Rosa26 reporter mice, X-Gal staining and double immunostaining for Cre and 5-HT revealed raphe nuclei-specific Cre expression. The efficiency of Cre recombination was assessed by breeding Pet1-Cre mice with carriers of a floxed Lmx1b gene. In the resulting mice, 5-HT and SERT were almost absent in the whole brain, except for a single 5-HT-positive neuron found in the dorsal raphe (DR) (1%). It was thereby shown that adult Lmx1bPet1-Cre mice lack essentially all 5-HT neurons and terminals. The HPLC measurements still revealed the presence of residual 5-HT, although at concentrations 10 fold lower than in wild type animals. It is unclear whether this is due to undetected serotonergic neurons that did not undergo recombination or the HPLC results reflect contamination by blood 5-HT due to insufficient perfusion with saline before tissue isolation. Information about non-serotonergic Cre expression was not provided for this line.

2.2.5. BAC-Pet1-CreERT2 The same group also generated a BAC-Pet1-CreERT2 transgenic mouse line that allows TMX-inducible recombination.68 In this model, recombination was shown to be specific for serotonergic neurons in the DR and raphe magnus (RM), but only 85% of TPH2 expressing neurons co-expressed the floxed reporter gene after TMX treatment. Crossbreeding of this Pet1-CreERT2 line with Lmx1bfl/fl mice led to 40% reductions in brain 5-HT levels and downregulation of serotonergic markers.68 One limitation of this mouse line is its FVB/N background used for the generation of founder lines. Most floxed mice are generated on a C57BL/6 background, which has become the most widely used strain for developmental and behavioral studies. The FVB/N line was reported to have higher than C57BL/6 mouse line locomotor activity, anxiety, and basal body temperature and is homozygous for the Pde6brd1 allele, causing retinal degradation (http://jaxmice.jax.org/strain/001800.html). As a consequence, most FVB/N



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mice are blind69 and thus, not suitable for behavioral experiments. This phenotype might also be inherited after crossing the Pet1-CreERT2 line to other strains and might have an unpredictable impact on experimental results.

2.3. Cre recombinase expression driven by the Tph2 promoter 2.3.1. BAC-TPH2-CreERT2 After discovery of the brain specific TPH isoform6 the Tph2 promoter was used to direct Cre expression selectively to 5-HT neurons. In 2009, the group of D. Bartsch applied a BAC-based transgenic approach to create TMX-inducible TPH2-CreERT2 mice.70 Besides higher specificity of the Tph2 promoter for serotonergic neurons in comparison to SERT or Pet1, use of CreERT2 in this line allows recombination at any stage of development or adulthood. The same BAC-based construct was also later used to generate a TPH2CreERT2 rat line.71 High specificity of Cre expression upon TMX treatment was demonstrated in both TPH2-CreERT2 mouse and rat lines. However, rats showed a lower recombination efficacy compared to mice (77% in rats vs. 90% in mice) (Table 1).

2.3.2. BAC-TPH2-CreERT2 A comparable TPH2-CreERT2 mouse line, based on a BAC construct, was also generated by Yadav and colleagues72 to study leptin-dependent 5-HT control of appetite. Characterization of this line after breeding with Rosa26loxP-STOP-lacZ mice52 and TMX treatment revealed β-Gal staining in DR, median raphe (MnR), and caudal raphe (CR) nuclei of the brainstem but not in hypothalamic or other regions. The specificity for serotonergic neurons within these areas has not been determined however.

2.3.3. BAC-TPH2-iCreERT2 A similar mouse line (TPH2-iCreERT2) was independently generated by B. Gloss, (http://jaxmice.jax.org/strain/016584.html). However in this case, an improved version of Cre gene (iCre) was employed by applying mammalian codon usage and by lowering the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals.73, 74 We have confirmed iCreERT2-mediated recombination within the DR, but not in the other brain regions using crossbreeding of this mouse line to Rosa26loxP-STOP-lacZ,52 ROSA26loxP-stop-EYFP,53 and Rosa26loxP-STOP-tdTomato (Jackson Laboratories, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J Stock Number: 007914) reporter mice


Page 10 of 38

Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Fig.1 A, B, C). Moreover, injection of an adeno associated virus (AAV), EF1a-DIO-CheTAEYFP, into the medial DR indicated effective Cre-mediated recombination in TPH2iCreERT2 mice (Fig.1D). However, a more detailed analysis of the specificity and efficacy of Cre expression in this line is still pending.

In summary, a wide repertoire of genetic tools to target Cre-mediated recombination to serotonergic neurons is now available (Table 1). Pet1-Cre and SERT-Cre lines are well characterized and represent useful tools to study the serotonergic system, whereby Sertdriven Cre lines have higher specificity and recombination efficiency than Pet1-driven transgenes, as recently demonstrated in the comparative study of VMATSert-Cre and VMATPet1-Cre mice.75 However, both promoters are not exclusively selective for the serotonergic lineage, whereas Tph2-driven Cre expression represents a more specific tool and should be regarded as a better model to target 5-HT-producing neurons specifically in the future. Additionally, the recent availability of the Tph2-driven rat Cre line71 allowed researchers to apply the more sophisticated behavioral tests for higher cognitive functions that have been already developed for use in rats, but not in mice.

3. Targeting defined serotonergic subpopulations As described in the sections above, precise targeting of a transgene to most serotonergic neurons can be performed successfully using several different mouse lines, albeit with some limitations. However, the serotonergic system itself is heterogeneous and includes various

subpopulations

of

neurons

with

different

connectivity,

functions,

and

developmental origins. Manipulating these subpopulations selectively requires more advanced methods.

3.1 Dual recombinase strategies Making use of newly developed genetic technologies, the group of S. Dymecki, identified rhombomere (r)-defined 5-HT sublineages that differ in gene expression with respect to progenitor and precursor cells.66 These investigators have created a novel dual recombinase-responsive indicator allele, RC-Fela, and a Flpe recombinase driver line, ePet-Flpe. While RC-Fela serves

as

recombination reporter, ePet-Flpe directs

recombination to Pet1-expressing 5-HT precursors. Combining RC-Fela with ePet-Flpe and Cre lines, specific for particular rhombomeres, enables isolation and manipulation of specific 5-HT neuronal subpopulations arising from these rhombomeres. For example, En1-Cre mice expressing Cre under the engrailed 1 (En1) promoter were used to study the subpopulation of 5-HT neurons originating from r1. In a comparable approach, the ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

intersectional alleles RC-FrePe and RC-FPDi were used to identify 5-HT neurons mediating breathing reflex control.76 These triple transgenic animals provide new tools for in vivo manipulation of specific 5-HT subpopulations and for “functional mapping” studies. In another study using the same selective dual recombinase strategy, Kim and colleagues directed tetanus toxin light chain (tox) synthesis to serotonergic subpopulations and thereby inhibited vesicular neurotransmission in those neurons.77 By recombinase expression under Pet-1 and En1 promoters in En1-Cre, ePet1-Flpe, RC-PFtox mice, they targeted the subpopulation of Pet1-descendant 5-HT neurons arising from serotonergic progenitors in r1. Tox action was thereby restricted to cells expressing both Cre and Flpe recombinases and required two recombination events. Using the described dualrecombinase technique, higher specificity (targeting of individual serotonergic subgroups) was achieved when compared to single-recombinase or conventional transgenic methodologies.

3.2. Viral mediated gene transfer An alternative to transgenic approaches is virus-mediated gene transfer into specific brain structures. Spatial control of vector delivery can be achieved by using the appropriate stereotaxic injection coordinates. Adenoviruses were among the first viruses to be successfully used for the specific infection of DR.78 In this approach, stereotaxic injection of a Cre-expressing adenovirus (AdCre)79 into the DR of a Cre-dependent LacZ reporter mouse line CAG-LoxP-stopLacZ80 led to the expression of Cre recombinase in approximately 12% of serotonergic neurons. Within 9 days after the injection, 43% of Cre-expressing 5-HT neurons were also positive for LacZ, confirming the validity of the method to target Cre-recombination throughout the DR. Lentiviruses have also been applied to label serotonergic neurons. To drive the expression of the EGFP exclusively in serotonergic neurons, an optimized Tph2 promoter with a twostep transcriptional amplification design was employed.81 Stereotactic injections of such viruses to medulla oblongata covering some of the midline and ventral raphe led to EGFP expression that was 97% colocalized with TPH expression. Of particular importance is the ability of lentiviruses to stably integrate into the genome of non-dividing cells, which is useful for lineage tracing. Meanwhile more advanced techniques combining AAV and transgenic approaches have been developed to enable highly selective transgene expression. For example, by injecting a double-floxed AAV with an inverse open reading frame for a channelrhodopsin2ACS Paragon Plus Environment

Page 12 of 38

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mCherry fusion protein (AAV2 DIO ChR2-mCherry) into the raphe obscurus (RO) of ePetCre mice, DePuy and colleagues showed that roughly half of 5-HT neurons in the raphe area were infected and 97% of infected cells were serotonergic.82 Recently, sindbis virus, canine adenovirus serotype 2 (CAV2), and rabies viruses have been developed for anterograde, retrograde, and transsynaptic retrograde transport, respectively, and used to study serotonergic circuitry.83-90

3.2.1. Anterograde transport Suzuki et al. injected a GFP-tagged sindbis virus into the DR for anterograde tracing of single serotonergic neurons to the olfactory bulb.87 The advantage of using sindbis virus is its high transfection efficiency in the CNS and its capacity to carry large amounts of genetic material. However, this virus is suitable only for acute in vivo work as infected neurons start degenerating 7 days after viral injection.91

3.2.2. Retrograde transport For retrograde tracing studies, Wu et al. injected retrogradely transported CAV2-Cre virus into the Nucleus tractus solitarii (NTS) of conditional TPH2lox/lox mice.90 Upon transport from the injection site to the cell bodies, Cre inactivates the Tph2 gene, specifically in neurons with projections to the injected area. TPH2 staining was decreased in RO and RM serotonergic neurons by 60-80%, while DR neurons were unaffected. As a result, 5-HT levels in the NTS of injected TPH2lox/lox mice were decreased by about 75%. In combination with ablation of specific neuropeptide-expressing neurons, this study allowed elucidation of the serotonergic mechanisms of feeding and body weight regulation.90 Interestingly, many AAVs, though not all, show a robust capacity for retrograde infection and subsequent transgene expression in mice. To isolate neuromodulatory serotonergic neurons that send axons to the basolateral amygdala (BLA), Rothermel et al.85 used recombinant, pseudotyped AAVs (rAAVs) engineered to improve transduction efficiency and tissue tropism using capsid genes from other AAV serotypes (e.g., AAV2/1). The applied virus carried a Cre-dependent expression switch (2/9.DIO.ChR2-EYFP) to allow ChR2-EYFP expression only in Cre-positive cells. Stereotaxic injection into the BLA of SERT-Cre mice resulted in ChR2-EYFP expression in a number of raphe neurons, mainly in the DR.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.2.3. Transsynaptic transport To analyze monosynaptic inputs to DR and MnR serotonergic neurons, Ogawa and colleagues used a retrograde transsynaptic system based on a modified rabies virus that initially infects only cognate receptor (TVA protein) expressing cells and lacks a glycoprotein required for transsynaptic spread.83 SERT-Cre mice50 were injected with two helper viruses expressing the glycoprotein and TVA after Cre recombination into either DR or MnR.88 This allowed the virus to spread transsynaptically to cells with direct synaptic contact to DR or MnR serotonergic neurons. Here, 95.8 % of infected TVA expressing cells were shown to be serotonergic and 30.5 % of 5-HT neurons were infected and expressed TVA. A small number of neighboring 5-HT-containing neurons were targeted as well.83 A rabies virus tracing strategy in SERT-Cre mice was also used in other studies to generate a whole-brain atlas of putative monosynaptic inputs onto forebrain-projecting DR and MnR serotonergic neurons or to characterize the distinct excitatory, inhibitory, and peptidergic inputs to the DR.84,

86, 89

However, released rabies virus can be taken up by

adjacent terminals that may or may not be synaptically connected.92 Classification of connectivity patterns will permit further functional dissection of the serotonergic system and enable analysis of the physiological relevance of specific input circuits. Taken together, viral delivery entails some disadvantages in comparison to transgenic animals such as postoperative recovery, limited injection volumes, and challenges associated with injection accuracy. Moreover, adeno93-97 and sindbis91 viruses might evoke immune responses and inflammation. The regular serotypes (1-10) of AAV cause minimal in vivo immune response,98 but some may cause gliosis99 and neurological deficits. Lentiviruses are more conservative in this respect and pose less of a risk of CNS damage. As an advantage viruses permit much higher expression levels of transgenes and enable retrograde transport enabling labeling and manipulation of neuronal subpopulations defined by their axonal terminals. Furthermore, viral mediated transgene expression represents a useful tool to deliver light-sensitive proteins for optogenetic approaches (see below). Though conventional viral vectors cannot incorporate very large transcriptional control units similar to BACs, they are optimal for the delivery of small transgenes such as Cre recombinase or Cre-dependent expression cassettes. Due to the ease and low cost of viral production in comparison to transgenic animals, viral methodology allows facile implementation of new genetic tools into experimental designs.


Page 14 of 38

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. Temporal control of gene expression within the serotonergic system 4.1. Inducible Cre-expressing mouse lines Performing a genetic manipulation at a selected time point during the lifetime allows the study of age-specific biochemical and behavioral effects within the serotonergic system. Several inducible Cre lines that enable excision or activation of genes at chosen time points within the serotonergic system are described in the sections above49, 68, 70, 71, 100 and are summarized in Table 1. However, these are not without limitations. Recombination mediated by Cre or other recombinases is an irreversible and relatively slow process. Furthermore, repeated TMX injections are necessary to permit drug delivery to all serotonergic neurons and to induce appropriate recombination rates. While TMX is FDA (U.S. Food and Drug Administration) approved, administration at doses higher than 150 mg/kg has been shown to evoke toxic effects in mice.101, 102 Moreover, it should be taken into account that TMX is a strong estrogen receptor antagonist. Therefore, minimal doses of TMX should be used. Moreover, recent studies demonstrated that systemic activation of CreERT2 might elicit hematological toxicity.101, 102 Therefore, use of proper controls, such as Cre+GOIwt/wt + TMX is essential.

4.2 Tetracycline-sensitive systems Audero et al. suppressed serotonergic firing by overexpressing 5-HT1A autoreceptors reversibly in transgenic mice,103 using the tetracycline-Off (tet-Off) system.104,

105

Double

transgenic mice were generated by breeding mice carrying the tet-promoter (Ptet) inserted upstream of the endogenous 5-HT1A receptor gene (5-Htr1atetO allele) with mice encoding the tetracycline transactivator (tTA) inserted downstream the ATG codon of the endogenous Slc6A4 (gene coding for SERT). tTA is an artificial transcription factor, that binds to Ptet, which in turn, mediates the overexpression of a GOI, in this case the 5Htr1a. Administration of Doxycycline (Dox, a stable tetracycline analogue) in this model leads to the prevention of transactivation of 5-HT1A receptors and, therefore, allows temporal control of their expression. Although 5-HT specificity is based on the Sert promoter, which might be also active in non-serotonergic cells, 5-HT1A receptor overexpression in double-transgenic animals is restricted to raphe nuclei of the mid- and hindbrain. However, it has not been determined whether the affected cells are all serotonergic. ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

Weber et al. designed a comparable ON/OFF system for genetic manipulations in 5-HT neurons.106 Similar to Audero and colleagues, they generated double-transgenic mice encoding tTA under the Tph2 promoter and a nLacZ reporter under the control of the-tetresponse

system

(TPH2-tTA/Ptet-nLacZ

mice).

Dual-label

fluorescence

immunohistochemistry identified LacZ expressing cells as serotonergic. Extraserotonergic brain regions were largely devoid of X-Gal staining, except for sparse β-Gal activity in the hypothalamus and beneath the aqueduct. Additionally, in the raphe, a few TPH2-negative cells were β-Gal positive, revealing some nonspecific gene targeting. Dox administration completely suppressed Ptet-mediated reporter expression and subsequent Dox withdrawal resulted in reactivation. When 5-HT-specific tTA mice are mated with Ptet-Cre/loxP-flanked target gene animals, spatially controlled, inducible gene deletion (TPH2-tTA/Ptet-Cre/loxP) can be achieved, comparable to TMX-inducible CreERT2 systems. In addition to the spatial control by the chosen promoter, temporal control of gene expression is possible with the described tetOff methodologies. Unlike the Cre/loxP system, gene manipulations are reversible. However, Ptet-controlled gene expression after Dox withdrawal occurs with a delay, depending on the animal’s age, Dox concentration, duration of treatment, and many other factors that have to be controlled for. In contrast to i.p. administration of TMX, Dox treatment is more suitable for behavior studies as it can be added to the drinking water diminishing effect of stress. Since Dox has a bitter taste, sugar or sweeteners are often added to Dox solutions that may lead to an increase in drinking volume, additional body weight gain, or other unexpected behavioral outcomes.

4.3 DREADDs A different method for efficient modulation of signal transduction in selected neurons is the introduction of Designer Receptors Exclusively Activated by Designer Drugs (DREADD) – engineered G-protein-coupled receptors that are activated by otherwise inert synthetic ligands. During recent years, several modifications of this technique have been used for in vitro and in vivo experiments.107-109 The example below made use of a Gi-DREADD (Di), a synthetic Gi/o-protein–coupled receptor that activates inwardly rectifying potassium channels upon stimulation with the compound clozapine-N-oxide (CNO) resulting in hyperpolarization and transient neuronal silencing.110 To study the influence of serotonergic activity on homeostatic control, Di was expressed via previously described dual-recombinase methodology with cell-subtype precision.66,


77,

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


109

A combination of the single recombinase-responsive allele RC-PDi, responsive to Cre,

with SERT-Cre was used to express Di in nearly all serotonergic neurons. These cells responded to CNO with hyperpolarization that led to the suppression of cell excitability. Furthermore, incorporation of a Cre-inducible reporter allele, RC-rePe, allowed visualization of Di expressing cells by GFP. Beyond serotonergic neurons, a population of extraserotonergic neurons in the thalamus was shown to express Di as well, due to presence of SERT-positive cells in this brain region. Current-clamp membrane potential recordings from cultured serotonergic neurons of the lower brainstem revealed a ~40 % reversible reduction in firing rates in Di expressing neurons after CNO exposure. Administration of CNO to transgenic animals resulted in rapid attenuation of chemoreflex, as well as a decrease in core body temperature representing vital physiological functions of 5-HT.109 DREADDs enable ligand-inducible inhibition of SERT-expressing neurons that is reversible within hours, and serves as model for functional mapping of neurons. CNOmediated inhibition happens within a shorter time frame as compared to Dox- or TMXinducible systems. At the same time, inertness of CNO has been questioned by others due to its redox partners and their biological effects.111 If CNO is not effectively eliminated by the kidneys, it can be reduced to the tertiary amine clozapine in vivo.112, 113 Various tertiary amine reduction routes generate compounds that efficiently act as agonists for DREADDs and a number of other receptors. In turn, receptor-mediated intracellular signaling pathways may be activated and impact animal physiology.114 These other possible actions of CNO metabolites are capable of reducing designer receptor–based cell and effect specificity and should be excluded by the use of appropriate controls. As another word of caution, all G-protein coupled receptors when artificially expressed at high levels are able to produce downstream coupling events without ligand presence. Various groups, including ourselves have observed this with DREADDs (S. Kasparov, unpublished observations). Therefore, it is critical to control for changes in cellular biochemistry or physiology in animals expressing DREADDs before CNO application.

4.4. Optogenetics Optogenetics is another current technique to modulate neuronal function that does not involve chemical ligands and enables much higher temporal resolution.115 Optogenetics enables researchers to manipulate firing rates of specific neuronal populations and to measure the outcome using physiological and behavioral readouts in vivo. For tight optical control of neuronal excitability, light-sensitive channels or light-driven ion pumps are expressed in genetically defined neurons. The most commonly used optogenetic actuator



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

is the non-selective cation channel channelrhodopsin, ChR2, and its derivatives. Using BAC technology, Zhao and colleagues generated a mouse line expressing ChR2 driven by the Tph2 promoter, TPH2-ChR2-EYFP.116 Co-localization of the reporter EYFP with 5-HT was shown for serotonergic neurons in the DR (116 and Fig.2A). Transgene expression was moderate in MnR and DR as approximately 15% of TPH2-positive cells were EYFPnegative.116 Electrophysiological recordings from the EYFP-positive neurons showed responsiveness to blue light. It is important to stress that ChR2 and similar proteins have to be expressed in the target cells at fairly high levels to enable efficient control of excitability. We employed this mouse model to study the impact of descending 5-HT-ergic control on sensory transmission in the spinal cord, based on previous studies that revealed a dual role for 5-HT in inhibition and facilitation of nociceptive input.117-120 These earlier studies relied mainly on gross lesions, broadly acting pharmacological manipulations, and electrical stimulation of the DR, techniques with obvious limitations such as compensatory re-wiring of the pathways, impact on the release of other neuromodulators,121, 122 and coactivation of non-5-HT neurons and fibres of passage.123 In theory, optogenetic stimulation of DR 5-HT neurons should help to overcome these limitations.124 In anaesthetized TPH2ChR-EYFP mice, optogenetic stimulation of 5-HT neurons in dorsolateral DR (coordinates from lambda: 0.3 mm ML, -1 mm AP, -3.4 mm DV; blue light stimulation: 10 msec ON, 10 msec OFF, 20 sec, 20 mV) did not significantly alter the expression of c-Fos, a marker of neuronal excitability,125 in the dorsal horn of the spinal cord (Fig. 2B), which serves as an index of the nociceptive input.126 Despite the refinement of the targeting techniques to specifically excite DR 5-HT neurons, the impact of serotonergic innervation of the spinal cord on nociceptive transmission remains ambiguous. Our negative finding might also indicate that different 5-HT subpopulations (that we could have stimulated simultaneously) have opposing effects on the nociceptive control so as to cancel each other out. To refine the spatial specificity, mouse lines expressing Cre recombinase under cellspecific promoters, in combination with viral delivery can also be used for optogenetic experiments. Such an approach was used in the study by Dugue et al., where viral vectors were injected into transgenic SERT-Cre mice (described above).127 In this study, response thresholds in awake behaving SERT-Cre mice following injection of viruses carrying Credependent ChR2-EYFP into the DR (coordinates from bregma: 0 mm ML, 4.4 to -4.7 mm AP, -2.8 to -2.9 mm DV, with a 340 caudal angle) were higher compared to control mice in the von Frey assay, a simple test of mechanosensitivity.128 The work of Dugue et al.127 and our studies (Fig. 2B-D) show that the response might differ between anesthetized and conscious animals, and that the roles of different serotonergic subpopulations may not be ACS Paragon Plus Environment

Page 18 of 38

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


identical. Delivery of light-sensitive channels is possible by combining the above described mouse lines expressing Cre under cell-type specific promoters with recently developed mouse lines expressing various opsins in a Cre-dependent manner.129 However, due to the heterogeneity of the serotonergic system, this will not allow precise targeting of particular subpopulations of 5-HT neurons. Finally, since rat transgenics are much less developed, injection of viral vectors with optogenetic actuators (either direct targeting with Tph2 promoter130 or a combination of TPH2-Cre with a Cre-dependent vector) may be used in this species. In any case, using blue light for activation of optogenetic constructs requires careful controls for the effects of temperature and blue light itself. For example, we recently reported that blue light induces activation-like signals in functional magnetic resonance imaging experiments probably due to the local heating.131 Control for the effects of blue light has become a particular issue since the recent demonstration of the vasomotor effects mediated by melanopsin in peripheral blood vessels.132 Development of more sensitive optogenetic tools that can be activated by application of low intensity light133-136 will help to overcome these concerns.

The techniques discussed in this section allow manipulations of specific neuronal populations in freely behaving animals with improved spatial and temporal precision. Optogenetics and DREADDs both involve the introduction of non-native proteins into neurons that thereby respond to defined stimuli (light or CNO) with altered excitability. For the analysis of temporal dynamics of specific circuits, optogenetics must be used, whereby an optical fiber needs to be implanted and manipulations can only affect the illuminated area in the brain. In contrast, by a simple systemic CNO administration all DREADDs distributed within large tissue volumes can be stimulated simultaneously. Likewise, the tetOff system allows gene expression control in all tissues expressing the chosen promotor. In all cases, unknown side effects of chemical agent administration or blue light have to be considered.101, 102, 131, 132, 137-139

Summary Serotonergic neurons in the raphe nuclei give rise to a complex and extensive network of axonal projections throughout the whole brain. A major challenge in the analysis of these circuits is to understand how the serotonergic networks are linked to the numerous ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

functions

of

this

neurotransmitter.

Several

new

methodologies

Page 20 of 38

to

manipulate

subpopulations of serotonergic neurons and to control 5-HT neuron activity have been developed during recent years, including the generation of different serotonergic-neuronspecific Cre lines, the invention of a dual recombination strategy, effective tools for temporal control of gene expression, DREADDs, and optogenetics. The nature of the experimental question dictates which option should be chosen as each has different merits and limitations. Importantly, since none of the described systems is totally free of nonspecific effects. For the most accurate and informative interpretation of results, adequate controls should be incorporated into experimental designs. Nonetheless, these new tools and methods, alone and in combination, enable powerful new routes to investigate the serotonin system.


Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgements: This work was supported by fellowship from the European Molecular Biology Organization (EMBO) (ASTF 72-2014) and in part by the grant from the Russian Science Foundation № 14-50-00069 to NA, and from the Federation of European Neuroscience (FENS) to VM (NENS, 2013).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abbreviations 5-HT - 5-hydroxytryptamine AAV - Adeno associated viral vector BAC - Bacterial artificial chromosome BLA - Basolateral amygdala CAV2 - canine adenovirus serotype 2 ChR2 - Channelrhodopsin 2 CNO - Clozapine-N-oxide CNS - Central nervous system CR - Caudal raphe Di - Synthetic Gi/o-protein coupled receptor (DREADD) Dox - Doxycycline DR - Dorsal raphe DREADD - Designer Receptors Exclusively Activated by Designer Drugs En1 - Engrailed 1 Flp – Flippase GOI - Gene of interest IHC - Immunohistochemistry LacZ - Gene coding for β-galactosidase Lmx1b - LIM homeobox transcription factor 1 NTS - Nucleus tractus solitarii MnR - Median raphe ObRb - Leptin receptor b LepRb - Leptin receptor b Pet1 - PC12 ETS domain-containing transcription factor 1 Ptet - Tet-promoter r - Rhombomere RM - Raphe magnus RO - Raphe obscurus SERT - Serotonin transporter SSRI - Serotonin reuptake inhibitor Tet - Tetracyclin TMX - Tamoxifen Tox - Tetanus toxin light chain TPH2 - Tryptophan hydroxylase 2 ACS Paragon Plus Environment

Page 22 of 38

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tTA - tetracycline transactivator VMAT - Vesicular monoamine transporter β-Gal - β-galactosidase DT - Diphteria toxin TVA protein - Cognate receptor



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

Figure legends Figure 1. Cre-mediated recombination in the TPH2-iCreERT2 mouse. A. LacZ expression in the raphe nuclei of the TPH2-iCreERT2 x R26R double transgenic mouse. DR – dorsal raphe nucleus, MnR – median raphe nuclei, CR – caudal raphe nuclei. B. EYFP expression (immunohistochemistry with anti-GFP antibody) in the dorsal raphe nuclei of the TPH2-iCreERT2 x ROSA26loxP-stop-EYFP double transgenic mouse. C. Expression of tdTomato in the raphe of TPH2-iCreERT2 x Rosa26loxP-STOP-tdTomato reporter mice. Arrowhead indicates neuronal cell bodies, arrow -indicates neuronal processes. D. EYFP expression (immunohistochemistry with anti-GFP antibody) in the dorsal raphe nuclei of the TPH2-iCreERT2 mouse injected with AAV virus (EF1a-DIOCheTA-EYFP). In all experiments Cre expression was induced by 3 consecutive TMX injections (120 mg/kg for A, B and C, and 100 mg/kg for D) on days 1, 3, and 5. Mice were sacrificed on day 12-15. Figure 2. TPH2-ChR2-EYFP mouse as a model for the study of serotonergic circuitry.

A.

Specificity

of

EYFP

expression

controlled

by

Tph2

promoter:

immunohistochemical analysis with anti-5-HT antibodies in dorsal raphe nuclei in TPH2ChR2 mouse (10x (left panel) and 40x (right panel) magnification. Neuronal activation in lamina I/II of the dorsal horn of the spinal cord was investigated through analysis of c-Fos expression (as previously described in

112

) following application of a series of noxious

pinch stimuli (pneumatic pincher: 2.5 N x 3) of the left hindpaw in control and TPH2-ChR2EYFP mice during optogenetic stimulation (10 msec ON, 10 msec OFF, 20 sec, 20 mV) in the dorsolateral dorsal raphe nucleus (coordinates from lambda: 0.3 mm ML, -1 mm AP, 3.4 mm DV). B. In both TPH2-ChR2 and control mice, higher numbers of c-Fos positive neurons were observed ipsilateral to the pinch stimulus compared to the contralateral side. No significant difference was observed between control and stimulated animals, however a large variance in the number of c-Fos positive neurons was observed in both groups of animals. n=8 per group. **p

Beyond Gene Inactivation: Evolution of Tools for Analysis of

Recommend Documents